Project Summary/Abstract Therapy for ischemic brain injury is poor in part because of our limited understanding of mechanisms leading to neuronal loss. While contributions of excessive glutamate release and neuronal Ca2+ accumulation have been much studied, recent evidence implicates critical contributions of another divalent cation, Zn2+. After ischemia or prolonged seizures, free Zn2+ accumulates in neurons, and observations that Zn2+ chelation is protective implicates a role in neuronal death. Culture studies have revealed that exogenously applied Zn2+ can enter neurons and accumulate in mitochondria, powerfully disrupting their function. However, little is known about mechanisms of injury caused by the accumulation of endogenous Zn2+ in native brain tissues. Using acute hippocampal slices subjected to oxygen glucose deprivation (OGD) to model ischemia, we recently made the first simultaneous measurements of cytosolic Zn2+ and Ca2+ changes, and found that Zn2+ accumulation is an early event in hippocampal pyramidal neurons, that precedes and contributes to subsequent cell death. These acute deleterious effects of Zn2+ appear to result specifically from Zn2+ uptake into mitochondria via the mitochondrial Ca2+ uniporter (MCU). The proposed studies are organized around a Hypothesis: Zn2+ contributes to the early stages of ischemic neuronal injury, in part via entering and inducing acute disruption of mitochondrial function. The primary goal of this study is to make an early assessment of this hypothesis in vivo, using of an established model of global cerebral ischemia resulting from asphyxial cardiac arrest. Aim I will use the cardiac arrest model to characterize Zn2+ and mitochondrial dependent changes and neuronal injury in different brain regions, as a function of both the duration of ischemia and the duration of recovery prior to assessment. Aim II will test a number of interventions targeting acute and subacute effects of Ca2+ and Zn2+, administered either alone or in combinations as suggested by our hippocampal slice studies, that we predict will abrogate certain shortcomings of the individual interventions. They will be delivered either before ischemia, or only upon reperfusion to assess potential for benefit with delayed administration. These studies will provide a bridge between compelling mechanistic clues derived from in vitro studies and efforts to forge better therapies for degeneration after in vivo ischemia, and we hope that they will lead to the elucidation of new types of interventions, to be delivered either during the acute phase of ischemia or upon reperfusion, that will disrupt the pathological cascade, enabling improved outcomes.